Abstract

Iproniazid (1-isonicotinoyl-2-isopropylhydrazine), an antidepressant drug removed from clinical use because of hepatic injury, and isopropylhydrazine, a metabolite of iproniazid, were found to be potent hepatotoxins in rats. This animal model was used in studies in vivo and in vitro to define better the biochemical and chemical mechanism(s) by which iproniazid and isopropylhydrazine mediate hepatotoxicity. Phenobarbital, an inducer of a class of hepatic microsomal cytochrome P-450 enzymes, greatly potentiated the necrosis, whereas inhibitors of these microsomal enzymes such as cobalt chloride, piperonyl butoxide and alpha-naphthylisothiocyanate, prevented the necrosis. Bis-para-nitrophenyl phosphate, an inhibitor of esterase and amidase enzymes, prevented the necrosis caused by iproniazid but had no effect on the necrosis caused by isopropylhydrazine. Iproniazid and isopropylhydrazine labeled with tritium or carbon-14 in the isopropyl group were found to bind covalently to hepatic tissue macromolecules, and those pretreatments that increased hepatic necrosis significantly increased covalent binding, whereas those pretreatments which prevented necrosis significantly decreased covalent binding. Iproniazid labeled with tritium in the pyridine ring or carbon-14 in the carbonyl group did not bind significantly to hepatic tissue. Rats that were given iproniazid or isopropylhydrazine, labeled specifically with tritium and carbon-14 on the c-2 methine position of the isopropyl group, expired acetone and carbon dioxide labeled with carbon-14. More importantly, propane was expired and contained a ratio of 3H/14C that was identical to that in the administered iproniazid or isopropylhydrazine and also identical to the 3H/14C ratio of the metabolite that was covalently bound to hepatic tissue macromolecules. Experiments carried out with rat liver microsomes and isopropylhydrazine specifically labeled with deuterium, tritium and carbon-14 support the view that isopropylhydrazine is the metabolite of iproniazid that is oxidized by a microsomal P-450 enzyme to a species that alkylates tissue macromolecules. Some of the urinary metabolites excreted by rats that were administered hepatotoxic doses of iproniazid and isopropylhydrazine have been identified by cochromatography and isotope dilution with synthetic standards and by comparative mass spectra. Compounds excreted into the urine of rats dosed with iproniazid include iproniazid, iproniazid-1-oxide, isonicotinic acid, isonicotinoyl glycine, acetylisoniazid, isopropylhydrazine, 1-acetyl-2-isopropylhydrazine and acetone. Isopropylhydrazine, 1-acetyl-2-isopropylhydrazine, and acetone have been found in the urine of animals administered toxic doses of isopropylhydrazine.